Recent progress in genetic engineering has enabled large scale production of various enzymes at a low cost, and economical process using an enzymatic reaction has also been enabled for the production of physiologically active substances of value that has traditionally been produced by means of bioconversion using a live bacterium, fermentation, or chemical synthesis.
In the meanwhile, an enzymatic reaction requiring energy as in the case of phosphorylation and amination needs adenosine 5′-triphosphate (ATP) for its energy donor or phosphate donor. In the conventional microbial transformation and fermentation, ATP has been supplied by the microorganism employed, whereas, in the case of the enzymatic process, addition of ATP to the reaction system and development of efficient ATP regeneration system are required.
However, process of inexpensive ATP synthesis has not yet been established and commercially available ATP is still very expensive. In addition, both the substrate and the enzyme used in the common ATP regeneration system, namely, the combination of phosphocreatine and phosphocreatine kinase, or acetyl phosphate and acetate kinase are very expensive, and their use has been unpractical and limited to the laboratory level.
In contrast to such high price of the ATP, adenosine 5′-monophosphate (AMP) can be produced at a relatively low cost. ATP is currently produced either by chemical synthesis or by using microorganism or yeast from AMP or adenine. Accordingly, development of an efficient ATP regeneration process has been highly awaited that can be used in the enzymatic reaction system using the ATP wherein the ATP is enzymatically produced from the relatively inexpensive AMP and the consumed ATP is efficiently regenerated instead of adding the expensive ATP.
In constructing a practical ATP generation/regeneration system, selection of the phosphate donor used is also important, and polyphosphate, which is inexpensive and stable, has been considered the most promising candidate of the phosphate donor. Enzymes which are known to be involved in the metabolism of the polyphosphate and which also act with adenosine nucleotide include polyphosphate kinase and polyphosphate:AMP phosphotransferase (hereinafter abbreviated as “PAP”).
PAP is an enzyme which phosphorylates AMP to produce ADP by using polyphosphate as the phosphate donor (J. Bacteriol., 173, 6484-6488(1991)). Zenhder et al. has reported that an ATP generation/regeneration system wherein AMP and polyphosphate are the substrates functions, when PAP obtained from Acinetobacter johnsonii is partially purified, and the partially purified PAP is used in combination with adenylate kinase (Appl. Environ. Microbiol., 66, 2045-2051(2000)). Kameda et al. has reported that, in the enzymatic reaction system wherein ATP is consumed to generate AMP, a system wherein ATP is generated from AMP using polyphosphate as the phosphate donor functions efficiently when the combination of the PAP from Myxococcus xanthus and E. coli polyphosphate kinase is used.
However, PAP is present in the Acinetobacter johnsonii cell in an extremely small amount, and with regard to the use of the crude PAP such as cell extract, a problem has been pointed out that contamination of the enzymes which decompose the substance involved in the reaction (AMP, ADP, ATP, reaction substrate and/or reaction product) may invite loss of reaction efficiency. Such problem can be obviated by the use of highly purified PAP instead of the crude enzyme. PAP, however, is very unstable, and the purification procedure of PAP is far too complicated to adopt such purified PAP into practical use.
In coping with such problem, the inventors of the present invention estimated that such problem can be challenged by producing the PAP of Acinetobacter johnsonii in a large amount using a recombinant DNA technique. However, the amino acid sequence of the enzyme and the gene for such enzyme have not been reported at all.